Tissue in vertebrates (in particular mammals) responds to cell or organ damage associated with an injury or a disease or aging by activating the regenerating system to recover from the cell or organ damage. Stem cells (tissue stem cells, somatic stem cells) that reside in the tissue play a significant role in this response. Stem cells have multipotency to differentiate into any cell or organ and are considered to compensate the cell or organ damage with this property to achieve the recovery. The regenerative medicine, the next-generation medicine that employs such stem cells, has raised high expectations.
The tissue for which stem cell studies are most advanced in mammal is bone marrow. It has been revealed that bone marrow contains hematopoietic stem cells of the living body and is the source of reproduction of all hemocytes. Furthermore, it has been reported that bone marrow contains stem cells that can differentiate into organs and tissues (e.g., bone, cartilage, muscle, fat, and the like), besides hematopoietic stem cells (see Non-Patent Document 1).
Recently, it has been further revealed that stem cells reside in every organ and tissue, such as skin, liver, pancreas, and fat, besides bone marrow and are responsible for the reproduction of each organ or tissue and maintenance of homeostasis (see Non-Patent Document 2 to 5). Moreover, stem cells that reside in organs and tissues have excellent plasticity and may be available in the reproduction of an organ or tissue whose self-replication has been impossible so far.
On the other hand, some of these stem cells have been known to decrease with aging and techniques for preventing the decrease of stem cells for maintaining homeostasis of the tissues have been actively studied (Non-Patent Document 6). Recently, techniques for culturing stem cells after the separation from living tissue for the proliferation thereof have been also developed in the fields of cell transplantation therapy and tissue engineering (regenerative medicine and regenerative cosmetic treatment) to apply the ability (multipotency) of stem cells to the reproduction of an organ or tissue (Non-Patent Documents 7 and 8).
In particular, it is very important for culturing stem cells in vitro to proliferate the stem cells while maintaining a state in which multipotency, the ability of stem cells, is maintained, that is, an undifferentiated state. The induction of differentiation by failing to maintain the undifferentiated state of the stem cells during the culture will result in the loss of the ability (multipotency) of the stem cells finally prepared and the failure to achieve the effect of interest (reproduction of an organ or tissue, or the like).
Based on the foregoing, it must be possible to culture stem cells while maintaining the undifferentiated state in order to use stem cells in cell transplantation therapy and tissue engineering (regenerative medicine and regenerative cosmetic treatment) for reproduction of an organ or tissue.
To date, some techniques for proliferating stem cells while maintaining the undifferentiated state have been reported, but such techniques are yet developing. For example, embryonic stem cells (ES cells) and hematopoietic stem cells can be maintained in an undifferentiated state by coculturing them with sustaining cells (stromal or feeder cells) (see Patent Document 1 and Non-Patent Documents 9 to 11). However, cases of infection with endogenous virus derived from feeder cells transmitted between different species of animals have been reported recently (cf. Non-Patent Document 12) and culturing stem cells using sustaining cells is not suitable for the culture of stem cells for medical applications.
Other methods include a method for maintaining the undifferentiated state of stem cells by combining cytokines complicatedly. For example, murine ES cells can be maintained in an undifferentiated state by adding LIF (Leukemia Inhibitory Factor)) to the medium (see Patent Document 2 and Non-Patent Document 13). In addition, the undifferentiated state has been reported to be maintained in the presence of early acting cytokines thrombopoietin (TPO), interleukin-6 (IL-6), FLT-3 ligand, and Stem Cell Factor (SCF) in embryonic stem cells, somatic stem cells, and the like (see Patent Document 3 and Non-Patent Document 14).
However, cytokines are expensive, problematic in harvesting their sources and storing, and difficult to be used in an easy way. In addition, it has been revealed that the effect of LIF is limited to very specific cell lines and ES cells and somatic stem cells in primate, in particular, cannot be maintained in an undifferentiated state only by the addition of LIF (see Non-Patent Document 10).
As seen above, currently reported methods for maintaining the undifferentiated state of stem cells require complicated manipulation and are not effective in maintaining the undifferentiated state. Therefore, a technique for proliferating stem cells while maintaining the undifferentiated state has been desired to use stem cells for regenerative medicine. Accordingly, a technique for proliferating stem cells safely, easily, and effectively while maintaining the undifferentiated state been desired.
Recently, patients with chronic wound such as diabetic foot ulcer or decubitus ulcer, which are hard to be treated only by epidermization have been increasing with increase of diabetes and arteriosclerosis and aging. Usually, a wound in the skin is treated with first aid such as washing of the wound and allowed to cure spontaneously by the healing ability of the living body. However, a wound healing is sometimes required to be promoted since natural healing may take a long time depending on the severity of the wound and elderly people and diabetic patients are in particular slow in natural healing in comparison with young people. As agents for promoting a wound healing in the skin, lysozyme chloride, solcoseryl, and the like are known but any of them has been hardly sufficiently effective in promoting a wound healing. In the wound healing of skin, tissue is reconstructed and therefore the migration of required cells and the production of extracellular matrices such as collagen are promoted. Type 3 collagen, which is mainly produced early in wound healing and is important for the reconstruction of tissue, has a promoting effect on wound healing and is used as an agent for promoting a wound healing (Non-Patent Document 15 and Patent Document 4). Moreover, as materials that stimulate the proliferation or differentiation of stem cells and promote a wound healing, substance P (Patent Document 5) and the like are known. However, these substances are problematic in cost and storage stability because of being protein and not suited for the case where a large amount of skin regenerative treatment in a short period of time, such as therapy application is required. Moreover, increased awareness of cosmetic treatment and anti-aging has heightened interest in regenerative cosmetic treatments of skin for improvement of wrinkles, slackness and pigmentation caused by aging and ultraviolet rays. Therefore, a substance that can be obtained easily, safe, and inexpensive is desired for skin regenerative medicine such as wound healing or regenerative cosmetic treatment of skin.